The work described in this proposal is aimed at understanding the mechanism and evolution of gene expression in kinetoplastid organisms, with special attention to antigenic variation and Trypanosoma brucei, the causative agent of African sleeping sickness. Studies on this organism are important not only because of the devastating disease it causes but also because of the unusual bio-chemistry and molecular biology which have been uncovered through its analysis. Many of these findings have been found to extend throughout the Kinetoplastida which includes other important pathogens such as T. cruzi and Leishmania. The first set of experiments described deal with antigenic variation in T.brucei. We will continue a gene family which comprises 9 highly conserved members encoding variant surface glycoproteins (VSGs) related to VSG117. Alteration in expression of VSGs is the means by which antigenic variation is effected by the parasite. Through detailed analysis of their genes, we will determine the structural relatedness of these antigens, the likely pathway of their evolution, and factors contributing to their different probabilities of expression. One of the most unusual pathways to be discovered is that of polycistronic transcription and its attendant trans-splicing. This complex mode of gene expression is apparently used in the transcription of most, if not all, nuclear-encoded protein-encoding genes in all members of the Kinetoplastida and, as such, represents an excellent target in the hopes of discovering a potential drug target: Experiments are described that will determine the structure and regulation of promoters for protein-coding genes and directly demonstrate that polycistronic transcription is indeed operating in these organisms. This will be accomplished through transfection elements with one or more detector genes downstream. The promoters for expression of the procyclin (or PARP) and tubulin genes will be studied as these represent examples of a developmentally regulated gene, procyclin, and of genes transcribed by a conventional polymerase activity, the tubulin genes (the procyclin genes, like VSG genes are apparently transcribed by an unusual, amanitin-resistant RNA polymerase). The factors responsible will also be identified and characterized. In addition to revealing important and novel pathways in biology, the results of these experiments may reveal new targets for the control of African trypanosomiasis.